Reagents for detecting Alu elements in cell-free DNA (cfDNA)

ABSTRACT

Provided herein is the use of measurements of cell-free DNA, protein, and/or metabolite found in biofluid (e.g., urine) for identifying and treating organ injury. Provided herein are methods and compositions for monitoring, detecting, quantifying, and treating kidney injury in subjects suffering from or suspected of having an altered renal status by measuring amounts of cfDNA and one or more other markers, such as inflammation markers, apoptosis markers, protein, and DNA methylation.

PRIOR RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/376,919, filed on Jul. 15, 2021, which is a continuation of U.S.patent application Ser. No. 16/325,385, filed on Feb. 13, 2019, which isa US National Stage application of International Patent Application No.PCT/US2017/047372, filed on Aug. 17, 2017, which claims the benefit ofU.S. Provisional Application No. 62/376,299 filed on Aug. 17, 2016, eachof which is incorporated by reference herein in its entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 081906_1276045_SEQ_LST.txt, createdon Oct. 8, 2021, 1110 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference in its entirety for allpurposes.

BACKGROUND OF THE INVENTION

Twenty-six million Americans suffer from organ injuries, such as thoseassociated with chronic kidney disease (CKD), and organ transplantrejection and dysfunction, e.g., kidney transplant or lung transplant.Treatment of these injuries is very costly. For example, twenty-eightpercent of annual Medicare spending, $57.5 billion, is spent on treatingCKD. However, for many of these, there is no way to predict when theinjury is imminent until clinical symptoms emerge. A large proportion ofthe costs associated with these diseases are due to lack of earlydetection leading to more severe organ injury and requiring greater andmore expensive therapeutic intervention. Methods for earlier detectionof kidney pathologies would enable reductions in medical costs and moreeffective therapeutic intervention.

Traditionally, detection of organ injury, e.g., kidney injury, is basedon examination of biopsies, which is both costly and invasive. Morerecently, cell-free DNA (cfDNA) from dying cells has been discovered inhuman urine. Recent efforts have focused on testing of urine for cfDNAas a marker for allograft rejection rather than organ injury;furthermore, the technique has been limited to using PCR ornext-generation sequencing for the detection of donor-specific SNPs orthe measurement of Alu elements. These methods have limitations becausethey are limited to testing for kidney rejection, are expensive, both inconsumables and equipment, require relatively larger quantities of DNA,and are not high-throughput. Further, they may not be able to detectfragment lengths shorter than 150 bp as it has been found that, althoughwhole, unfragmented DNA was optimal for a qPCR based approach formeasuring Alu, there was a significant reduction (75% reduction) in DNAquantification for DNA at a fragment size of <150 bp (Sedlackova et al.,Biol. Proced. Online (2013) 15:5. doi:10.1186/1480-9222-15-5).

BRIEF SUMMARY OF THE INVENTION

A novel methodology for quantitative analysis of cell-free DNA inbiofluid, such as urine and bronchoalveolar lavage (BAL), is provided.Aspects described herein include a preservative cocktail of reagentsthat can stabilize biofluid cellular DNA, a method of measuring cfDNA byhybridization assay that quantifies human Alu repeats in the biofluid,and a novel analysis method that factors in clinical assay variables toprovide a quantitative risk score for kidney injury. In some cases, theamounts of additional markers in the sample are measured, such asmethylation markers (e.g., 5-methylcytosine), tissue inflammationmarkers (e.g., CXCL10), apoptosis markers (e.g., kidney tubular injurymarkers, such as clusterin), total protein, and/or creatinine. Theseaspects can be used to assess the kidney health in the context of kidneytransplantation and kidney disease.

In one aspect, this disclosure provides a solution, e.g., a sterilesolution, that comprises a formaldehyde donor, a chelator,aurintricarboxylic acid, and polyethylene glycol (PEG) in aconcentration sufficient to inhibit cell lysis and to inhibit nucleasesin urine. In some embodiments, the solution further comprises sodiumazide and/or a buffer. In some embodiments, the solution furthercomprises a biofluidic sample. In some embodiments, the biofluidicsample is a urine sample or a BAL sample, as the effluent of choice fornon-invasive measurment of kidney or lung organ injury. In someembodiments, the urine sample is from a patient who has received akidney transplant or has acute or chronic kidney injury, in others it isthe bronchoalveolar lavage (BAL) fluid from a patient who has received alung transplant or has lung injury.

In one aspect, this disclosure provides a method of detecting Alu copynumber in a biofluid sample, the method comprising: obtaining a urine orother biofluid sample from a human, extracting cfDNA from the sample,forming a reaction mixture by contacting the cfDNA with a nucleic acidprobe under conditions to allow the probe to hybridize to DNA in thecfDNA that is complementary to the probe, wherein the nucleic acid probehas a nucleic acid sequence having a 3′ and 5′ end, wherein the nucleicacid probe is complementary to contiguous 20-292 nucleotides of SEQ IDNO:1, wherein the 3′ or 5′ end is covalently linked to a detectablelabel; and quantifying the amount of DNA hybridized to the probe. Insome embodiments, the probe is complementary to at least 50 contiguousnucleotides of SEQ ID NO:2. In some embodiments, the probe comprises asequence of 50-150, 70-100, 80-90, or exactly 81 nucleotidescomplementary to SEQ ID NO:1. In some embodiments, the detectable labelis biotin and the method comprises contacting the detectable label witha streptavidin-linked signal producing agent.

In some embodiments, before forming the reaction mixture, mixing asolution comprising diazolidinyl urea, ethylenediaminetetraacetic acid(EDTA), aurintricarboxylic acid, and polyethylene glycol (PEG) in aconcentration sufficient to inhibit cell lysis and to inhibit nucleasesinto the urine sample. In some embodiments, the method further comprisesquantifying the amount of creatinine in the reaction mixture. In someembodiments, the method further comprises normalizing the amount ofcfDNA hybridized to the probe against the amount of creatinine in theurine sample to produce a normalized amount of cfDNA. In someembodiments, the method further comprises normalizing the amount ofcfDNA hybridized to the probe against the amount of creatinine in theurine sample to produce a normalized amount of hybridized DNA. In someembodiments, the method further comprises determining the patient haskidney injury if the detected amount of target DNA (e.g., cfDNA)hybridized to the probe or the normalized amount of target DNA (e.g.cfDNA) is greater than the cutoff value.

In some embodiments, the human is a patient who has received a kidney orlung transplant and the presence of kidney or lung injury indicates thepatient may have acute rejection episodes. In some embodiments, themethod further comprises producing a prediction score for determiningkidney or lung health based on the normalized amount of target DNA(e.g., cfDNA) and the time post-transplant of the kidney. The patient isdetermined to have acute rejection episodes when the predictive score isgreater than a cutoff value for the predictive scores. In someembodiments, the urine or BAL sample is taken 0-400 days, or 10-100days, or 20-50 days from the patient's receiving a kidney or lungtransplant.

In some embodiments, the urine sample is from an individual suspected ofhaving a kidney injury caused by a disease selected from the groupconsisting of BK viral nephritis, focal segmental glomerulosclerosis(FSGS), kidney stone, acute tubular necrosis (ATN), IgA nephropathy(IgAN), and diabetic kidney disease or kidney disease from systemicdiseases such as hypertension and autoimmune disorders (eg SLY,rheumatoid arthritis). In these embodiments, a determination of kidneyinjury indicates the patient has the disease.

In some embodiments, the method further comprises quantifying the amountCXCL10 in the reaction mixture to add greater specificity andsensitivity to the assay through the addition of a biomarker reflectingthe inflammatory burden. In some embodiments, the method furthercomprises quantifying the proportion or absolute quantity of methylatedcfDNA and hydroxymethylated cfDNA to reflect the methylation status ofthe circulating DNA which further defines the presence of intrinsictissue injury.

In another aspect, the disclosure provides a reaction mixture comprisingnon-amplified cell-free DNA (cfDNA) extracted from a urine sample (e.g.,in some embodiments, from a patient who has received a kidneytransplant) and a nucleic acid probe having a nucleic acid sequence andhaving a 3′ and 5′ end, wherein the nucleic acid probe is complementaryto 20-292 contiguous nucleotides of SEQ ID NO:1 and wherein the 3′ or 5′end is covalently linked to a detectable label. The reaction mixture cancomprise any of the components described herein.

In another aspect, provided herein is a method of detecting Alu copynumber in cell-free DNA (cfDNA) in a biofluid sample from an individualhaving a lung transplant or lung transplant clinical conditions. Themethod comprises obtaining a biofluid sample, e.g., a bronchoalveolarlavage (BAL) fluid sample, from a human; extracting cfDNA from thebiofluidic sample; forming a reaction mixture by contacting the cfDNAwith a nucleic acid probe under conditions to allow the probe tohybridize to DNA in the cfDNA that is complementary to the probe,wherein the nucleic acid probe has a nucleic acid sequence having a 3′and 5′ end, wherein the nucleic acid probe is complementary tocontiguous 20-292 nucleotides of SEQ ID NO:1 and wherein the 3′ or 5′end is covalently linked detectable label; and quantifying the amount ofDNA hybridized to the probe, thereby detecting Alu copy number incell-free DNA (cfDNA) in the biofluidic sample. In some embodiments, thebiofluidic sample is a bronchoalveolar lavage (BAL) fluid sample andwherein the method further comprises quantifying the total volume of BALfluid.

In some embodiments, the method further comprises normalizing the amountof cfDNA hybridized to the probe against the total BAL fluid samplevolume to produce a normalized amount of hybridized cfDNA.

In some embodiments, the method further comprises comparing thenormalized amount of hybridized DNA to a cutoff value indicative of lungstatus.

In some embodiments, the method further comprises the determining thepatient has lung injury if the detected amount of target DNA (e.g.,cfDNA) hybridized to the probe or the normalized amount of target DNA(e.g., cfDNA) is greater than the cutoff value. In some embodiments, thehuman is a patient having received a lung transplant where in the lunginjury indicates that patient has rejection episodes.

In some embodiments, the method further comprises generating Kidneyinjury Test score, a KIT score, based on the amount of cfDNA hybridizedto the probe and the amount of creatinine in the reaction mixture. Insome embodiments, the KIT score is generated using the ratio of theamount of DNA hybridized to the probe to the amount of creatinine. Insome embodiments, the KIT score is generated by use of generalizedlinear models, such as logistic regression, using the amount of cfDNAhybridized to the probe, the amount of creatinine, one or moreinflammation markers (e.g., CXCL10), one or more apoptosis markers(e.g., kidney tubular injury marker such as clusterin), and/or one ormore DNA methylation markers present in the biofluid sample. In someembodiments, nonlinear regression models, selected from the groupconsisting of neural networks, generalized additive models, similarityleast squares, and recursive partitioning methods, may be used todevelop KIT Scores. In some embodiments, the KIT score is generatedusing additional biomarkers selected from the group of CXCL10, clusterinand DNA methylation markers such that the KIT score results in greatersensitivity and specificity for the diagnosis and prediction of organinjury, than the use of individual markers in the KIT assay. The KITinjury score also detects organ injury with greater sensitivity thancurrent markers of kidney function such as the serum creatinine or urineprotein or current markers of lung function, such as forced expiratoryvolume (“FEV”) and forced vital capacity (“FVC”). In some embodiments,using the IT score, e.g., a KIT score, as described above can detectkidney injury with a sensitivity of at least 85%, at least 87%, at least88%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, and/or a specificity of at least 85%, at least 87%,at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%. In some embodiments, using the KIT scoredescribed above can detect kidney injury with an AUC of at least 85%, atleast 87%, at least 88%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96.9%, or at least99.4%.

In some embodiments, the disclosure provides a method of detecting organinjury comprising measuring the amount of cfDNA, and amounts of one ormore of markers in a biofluid sample obtained from an organ that issuspected of having injury or is likely to develop injury, wherein oneor more markers is selected from the group consisting of: i) one or moreinflammation markers, one or more apoptosis markers, total protein, andiv) one or more of DNA methylation markers; producing an IT score usingthe amount of cfDNA and the amounts of the one or more markers, anddetermining the patient having injury in the organ or predict that thepatient will develop injury in the organ if the IT score is above apredetermined cutoff. In some embodiments, the organ injury is kidneyinjury. In some embodiments, the IT score is produced by furtherincluding creatinine. In some embodiments, the IT score is produced byusing a mathematical model using the amount of cfDNA hybridized to theprobe, the amount of creatinine, one or more inflammation markers (e.g.,CXCL10), one or more apoptosis markers (e.g., kidney tubular injurymarker such as clusterin), creatinine, and/or one or more DNAmethylation markers present in the biofluid sample

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show-electropherograms that demonstrate in the absenceof DNA preservative solution the DNA degrades and disintegratescompletely over a period of 72 hours; whereas DNA preservation solutionhelps maintain DNA integrity up to 72 hours. FIG. 1A shows the timecourse between 0 hours and 24 Hours. FIG. 1B shows the time coursebetween 48 hours and 72 Hours.

FIG. 2 compares using the methods disclosed herein and using thestandard PCR based methods in Alu detection for cfDNA analysis.

FIG. 3 shows a dual biotinylated-oligonucleotide complementary to theAlu repeats.

FIG. 4. shows the dynamic range of the assay disclosed herein having atleast 5 orders of magnitude using a chemiluminescent assay system.

FIGS. 5A-5D show using the assay disclosed herein to distinguish kidneytransplant rejection (FIGS. 5A-5B), native kidney disease (FIG. 5C) andits improved performance over proteinuria in detecting kidney injury.(FIG. 5D).

FIG. 6 shows the correlation between chromosome Y and chromosome 1 copynumber in the urine validates the method in this disclosure.

FIG. 7. shows an exponential decay curve showing change ofcfDNA/creatinine ratio over the number of days post-transplantationusing data from 9 patients who received kidney transplant and did notexperience acute rejection episodes.

FIG. 8A shows the change of cfDNA/creatinine ratio over the days ofpost-transplantation and FIG. 8B shows change of the Kidney injury Test(KIT) score over the days of post-transplantation.

FIGS. 9A and 9B show using the cfDNA/creatinine ratio to predict acuterejection in patient #2 and patient #3.

FIGS. 10A and 10B show using the methods disclosed herein to detect IgAnephropathy (a native kidney disease) in patients. In this study, urinesamples from individuals—117 of which were healthy and 85 individualshad IgA nephropathy, as previously determined by other methods—wereanalyzed using the methods disclosed herein. FIG. 10A shows thedifferences in the detected cfDNA/creatinine ratios between healthyindividuals (“normal”) and those having IgA nephropathy (“IgA”). The ROCanalysis indicates that the AUC of the ROC curve is 0.9651 with aconfidence interval of 0.9431 to 0.9871 with a P value of <0.0001. FIG.10B.

FIG. 11 shows a comparison between the cfDNA concentration found inbronchoalveolar lavage (BAL) fluid from individuals who had stable lungtransplants (“STA”) and from those who underwent occult, acute, orchronic rejection (“Rejection”). The p-value was 0.0427.

FIG. 12 shows a predictiveness curve for the probability of acute kidneyrejection in kidney transplant patients as a function of a KIT score,which is generated using measurements of cfDNA and creatinine fromurine. Urine samples from 41 patients having received kidney transplantswere collected as disclosed herein. Measurements of cfDNA and creatininein urine were obtained from each patient.

FIG. 13 shows using a KIT score to assess kidney injury based on adataset of 490 clinical samples with multiple causes of kidney injury.

FIGS. 14A-14E show using generalized linear model and associated ROCcurves to predict probability of kidney injury as well as variousdiseases that are associated with kidney injury, namely, type IIdiabetes mellitus, immune response, kidney stones, transplant rejection,and hypertension.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the agent” includes reference to one or more agents knownto those skilled in the art, and so forth.

The terms “subject”, “patient” or “individual” are used hereininterchangeably to refer to a human or animal. For example, the animalsubject may be a mammal, a primate (e.g., a monkey), a livestock animal(e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal(e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, aguinea pig, a bird), an animal of veterinary significance, or an animalof economic significance.

The term “nucleic acid”, or “polynucleotide” includesdeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. The term nucleic acid is used interchangeablywith gene, cDNA, and mRNA encoded by a gene.

The term “kidney injury status” refers to whether the patient showsinjury in the kidney. For purpose of this disclosure, kidney injury canresult from surgery, such as kidney transplant, or from any kidneydisease.

The term “kidney transplantation” or “kidney transplant” refers to theorgan transplant of a kidney into a patient. The source of the donorkidney can be from a deceased or living donor.

The term “Alu” or “Alu element” or “Alu repeat” refers to a shortstretch, about 300 base pairs long, of DNA originally characterized bythe action of the Arthrobacter luteus restriction endonuclease. Aluelements are the most abundant repetitive elements in the human genome.They are derived from the small cytoplasmic 7SL RNA. SEQ ID NO:1represents an exemplary human Alu repeat.

The term “hybridize” refers to the annealing of one or more probes to atarget nucleotide sequence. Hybridization conditions typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

The term “biofluid” or “biofluidic sample” refers to a fluidiccomposition that is obtained or derived from an individual that is to becharacterized and/or identified, for example based on physical,biochemical, chemical and/or physiological characteristics. Non-limitingexamples of biofluid include blood, serum, plasma, saliva, phlegm,gastric juices, semen, tears, and sweat. In one embodiment the biofluidis urine. In another embodiment the biofluid is BAL.

The term “post-transplantation” refers to a time after thetransplantation of an organ, e.g., kidney or lung, into the patient froma donor.

As used herein, the term “AUC” refers to “area under the curve” orC-statistic, which is examined within the scope of ROC(receiver-operating characteristic) curve analysis. AUC is an indicatorthat allows representation of the sensitivity and specificity of a test,assay, or method over the entire range of test (or assay) cut pointswith just a single value. An AUC of an assay is determined from adiagram in which the sensitivity of the assay on the ordinate is plottedagainst 1-specificity on the abscissa. A higher AUC indicates a higheraccuracy of the test; an AUC value of 1 means that all samples have beenassigned correctly (specificity and sensitivity of 1), an AUC value of50% means that the samples have been assigned with guesswork probabilityand the parameter thus has no significance.

Using AUCs through the ROC curve analysis to evaluate the accuracy of adiagnostic or prognostic test are well known in the art, for example, asdescribed in, Pepe et al., “Limitations of the Odds Ratio in Gauging thePerformance of a Diagnostic, Prognostic, or Screening Marker,” Am. J.Epidemiol 2004, 159 (9): 882-890, and “ROC Curve Analysis: An ExampleShowing The Relationships Among Serum Lipid And Apolipoprotein levels InIdentifying Subjects With Coronary Artery Disease,” Clin. Chem., 1992,38(8): 1425-1428. See also, CLSI Document EP24-A2: Assessment of theDiagnostic Accuracy of Laboratory Tests Using Receiver OperatingCharacteristic Curves; Approved Guideline—Second Edition. Clinical andLaboratory Standards Institute; 2011; CLSI Document I/LA21-A2: ClinicalEvaluation of Immunoassays; Approved Guideline—Second Edition. Clinicaland Laboratory Standards Institute; 2008.

As used herein, the term “diagnose” means assigning symptoms orphenomena to a disease or injury. For the purpose of this invention,diagnosis means determining the presence of organ injury in a subject.

As used herein, the term “predict” refers to predicting as to whetherorgan injury is likely to develop in a subject.

1. Kidney Injury Status

Compositions and methods are provided that can be used to assess kidneyinjury status, i.e., the presence or absence of kidney injury in anindividual. Such an assessment is helpful for diagnosing when anindividual is in need of medical intervention, such as being given moremedication to address the medical problem or having medication decreased(including cessation) where it is no longer medically necessary. Forexample, compositions and methods described herein can be used todetermine when an individual has kidney injury due to kidney transplantor kidney disease.

Kidney injury can develop in patients who have undergone a kidneytransplant. This can happen because of several immune and non-immunefactors such as ischemia reperfusion injury, size disparity, donorrelated factors, cell-mediated rejection, and antibody-mediatedrejection, by way of example. Problems after a transplant may include:transplant rejection (hyperacute, acute or chronic), infections andsepsis due to the immunosuppressant drugs that are required to decreaserisk of rejection, post-transplant lymphoproliferative disorder (a formof lymphoma due to the immune suppressants), imbalances in electrolytesincluding calcium and phosphate which can lead to bone problems amongother things, and other side effects of medications includinggastrointestinal inflammation and ulceration of the stomach andesophagus, hirsutism (excessive hair growth in a male-patterndistribution), hair loss, obesity, acne, diabetes mellitus type 2,hypercholesterolemia, and osteoporosis.

Kidney injury can also develop in patients having kidney disease. Kidneydiseases are diverse, but individuals with kidney disease frequentlydisplay characteristic clinical features. Common clinical conditionsinvolving the kidney include but are not limited to the nephritic andnephrotic syndromes, renal cysts, acute kidney injury, chronic kidneydisease, diabetes-induced nephropathy, urinary tract infection,nephrolithiasis, and urinary tract obstruction, glomerular nephritis(GN), focal segmental glomerular sclerosis (FSGS), IgA nephropathy(IgAN), mesangiocapillary, lupus and membranous etc, hypertensivenephropathy, and drug induced nephropathy. Kidney diseases can alsoinclude the various cancers of the kidney which exist. For example suchcancers include, but are not limited to, renal cell carcinoma,urothelial cell carcinoma of the renal pelvis, squamous cell carcinoma,juxtaglomerular cell tumor (reninoma), angiomyolipoma, renal oncocytoma,bellini duct carcinoma, clear-cell sarcoma of the kidney, mesoblasticnephroma, Wilms' tumor, mixed epithelial stromal tumors, clear celladenocarcinoma, transitional cell carcinoma, inverted papilloma, renallymphoma, teratoma, carcinosarcoma, and carcinoid tumor of the renalpelvis. Kidney disease can also be virally induced and include, but arenot limited to BKV nephropathy and nephropathy induced by EBV and CMV.Kidney disease can also be drug-induced as some medications arenephrotoxic (they have an elevated risk for harming the kidneys). In theworst case, the drug causes kidney failure, while in other cases, thekidneys are damaged, but do not fail. Common nephrotoxic drugs include,but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs),some antibiotics, some painkillers, and radiocontrast dyes used for someimaging procedures.

In some embodiments, a urine sample is from an individual having akidney transplant, or one of the above-listed kidney disorders or kidneytransplant clinical conditions described above is assayed as describedherein.

2. Lung Injury Status

Compositions and methods are provided herein that can be used to assesslung injury status, i.e., the presence or absence of lung injury in anindividual. Such an assessment is helpful for diagnosing when anindividual is in need of medical intervention, such as being given moremedication to address the medical problem or having medication decreased(including cessation) where it is no longer medically necessary. Forexample, the compositions and methods described herein can be used todetermine when an individual has lung injury due to lung transplant.

Lung injury can develop in patients who have undergone a lungtransplant. This can happen because of several immune and non-immunefactors such as ischemia reperfusion injury, size disparity,donor-related factors, cell-mediated rejection, and antibody-mediatedrejection, by way of example. Problems after lung transplantation mayinclude hyperacute rejection, acute rejection, several types of chronicrejection or chronic lung allograft dysfunction (CLAD) such asrestrictive allograft syndrome (RAS) or bronchiolitis obliteranssyndrome (BOS), infections, and sepsis due to the immunosuppressantdrugs that are required to decrease risk of rejection.

In some embodiments, a biofluid sample, e.g., a bronchoalveolar lavage(BAL) fluid sample, is from an individual having a lung transplant, orlung transplant clinical conditions described above is assayed asdescribed herein. Thus, in some embodiments, provided herein is a methodof detecting Alu copy number in cell-free DNA (cfDNA) in a biofluidsample from an individual having a lung transplant or lung transplantclinical conditions. The method comprises obtaining a biofluid sample,e.g., a bronchoalveolar lavage (BAL) fluid sample, from a human;extracting cfDNA from the biofluidic sample; forming a reaction mixtureby contacting the cfDNA with a nucleic acid probe under conditions toallow the probe to hybridize to DNA in the cfDNA that is complementaryto the probe, wherein the nucleic acid probe has a nucleic acid sequencehaving a 3′ and 5′ end, wherein the nucleic acid probe is complementaryto contiguous 20-292 nucleotides of SEQ ID NO:1 and wherein the 3′ or 5′end is covalently linked detectable label; and quantifying the amount ofDNA hybridized to the probe, thereby detecting Alu copy number incell-free DNA (cfDNA) in the biofluidic sample

The amount of DNA hybridized to the probe may be normalized against thetotal biofluid sample volume to produce a normalized amount ofhybridized DNA. In some cases, the normalized amount of hybridized DNAis compared to a cutoff value indicative of lung status. In some cases,the method further comprises determining the patient has lung injury ifthe detected amount of target DNA (e.g., cfDNA) hybridized to the probeor the normalized amount of target DNA (e.g., cfDNA) is greater than thecutoff value.

3. The Preservation Cocktail

In some aspects, a preservative cocktail of reagents that can stabilizebiofluid cell-free DNA (“cfDNA”) is provided. In some embodiments, thepreservative cocktail is a solution comprising a formaldehyde donor anda chelator, such as a calcium chelating agent. Non-limiting examples offormaldehyde donor include diazolidinyl urea and imidazolidinyl urea.Non-limiting examples of chelators include EDTA and EGTA. In someembodiments, the solution further comprises one or both of PEG,aurintricarboxylic acid. In some embodiments, the solution furthercomprises sodium azide, and a buffer. Non-limiting examples of buffersinclude PBS, TAPS, Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES,Cacodylate, and IVIES. In some embodiments, the solution comprises therespective components at a concentration that can preserve the biofluid(e.g., urine) by preventing degradation of cfDNA by nucleases, and/or byinhibiting cell lysis. In addition to stabilizing cfDNA, the solutioncan also prevent genomic DNA contamination through stabilisation ofcells present in the biofluid. Various molecular weight of PEG andvarious forms of EDTA can be used in this cocktail to provide thedesired properties above. In one embodiment, the PEG used in thecocktail is PEG 35,000. In one embodiment, EDTA is Na2EDTA. In oneembodiment, EDTA is K2EDTA. In one embodiment, EDTA is K3EDTA.

Typically, the cocktail is prepared as a concentrated stock solution,e.g., a 100×, 20×, 10×, 5×, or 2× concentrated stock solution, and ismixed and diluted with biofluid to be assayed, e.g., a urine sample, toa final working concentration. For example, for a 10× concentration ofthe cocktail, which will be diluted 10 times after mixing with urine,the concentration of diazolidinyl urea can be 0.1 g/L to 50 g/L, e.g.,0.5 g/L to 30 g/L, 1 g/L to 20 g/L, 5 g/L to 20 g/L, 5 g/L to 15 g/L, or5 g/L to 10 g/L; the concentration of PEG 35,0000 can be 0.2 g/L to 50g/L, e.g., 0.5 g/L to 40 g/L, 1 g/L to 30 g/L, 20 g/L, or 15 g/L to 25g/L; the concentration of aurintricarboxylic acid can be 0.1 mM to 10mM, e.g., 0.0.2 mM to 10 mM, 0.0.5 mM to 5 mM, 0.5 mM to 2 mM, or 1 mMto 2 mM. The concentration of EDTA can be 1 mM to 100 mM, e.g., 2 mM to50 m, 5 mM to 40 mM, 5 mM to 20 mM, or 10 mM to 20 mM. The 10×concentrated stock solution may also comprise sodium azide at aconcentration of 1 mM to 100 mM, 2 mM to 50 mM, 5 mM to 20 mM, or 10 mMto 15 mM. The cocktail can comprise a buffer, such as phosphate buffersaline (PBS), for example 10×PBS in a 10× concentrated stock solution.

In some cases, the cocktail is provided in a powder or solid tableformat and is reconstituted to solution by adding water or aqueousbuffer, or the biofluid itself.

In certain embodiments, the cocktail comprises about 10 g/L DiazolidinylUrea, about 20 g/L PolyEthelyene Glycocol, about 1 mM Aurintricarboxylicacid, about 10 mM K2EDTA, about 10 mM Sodium Azide, and/or about 1×Phosphate Buffered Saline, pH7.4.

4. The Nucleic Acid Probe

The Alu gene has been extensively studied for amplification of variousregions for assessing the quantity of cfDNA in patients with cancer(Biol Proced Online (2013); Park et al., Oncol Lett (2012)). In oneaspect, the present invention provides a method of assessing thequantity of cfDNA in biofluids by using a labeled nucleic acid probe tohybridize to the Alu repeats in cfDNA.

In some embodiments, the nucleic acid probe comprises a nucleotidesequence that comprises, or is fully complementary to, at least 20-300,e.g., 20-292, 20-180, 50-150, 70-100, 80-90 contiguous nucleotides ofSEQ ID NO: 1:

5′GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCA GCACTTTGGGAGGCCGAGGCGGGCGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGT GAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGCGCGCCTGTAATCCCAGCTAC TCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCGGAGGTTGCAGTGAGCCGAGATCGCGCCA CTGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAA-3′.

In some embodiments, the probe comprises a nucleotide sequence thatcomprises, or is fully complementary to, at least 20, 30, 40, 50, 60,70, 80, or 81 contiguous nucleotides of SEQ ID NO: 2:

GCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAG GAGAATCGCTTGAACCCGGGAGGCGGAGGTTGCAGTGAGCCGAGAT

A nucleotide referred to herein in the context of the nucleic acid probecan be a natural nucleotide, e.g., cytosine, guanine, thymine,adenosine, or a modified nucleotide. A modified nucleotide refers to analteration in which at least one nucleotide of the oligonucleotidesequence is replaced by a different nucleotide that provides a desiredproperty (e.g., ability to base pair with a complementary nucleotide ina target nucleic acid) to the oligonucleotide. Non-limiting examples ofmodified nucleotide include locked nucleic acids (LNA), peptide nucleicacids (PNA), morpholinos, and those described in US Pat. Pub. No.20160177377.

In some embodiments, the nucleotide sequence of the probe is conjugatedto a detectable label. The detectable label can be conjugated onto anynucleotide of the probe so long as it does not inhibit hybridization ofthe probe to the target, i.e., the Alu repeats in the cfDNA. In someembodiments, the 5′ end of the nucleotide sequence is conjugated to oneor more (e.g., two or more) detectable label. In some embodiments, thedetectable label itself is conjugated to the signal-producing agent. Insome embodiments, the detectable label is a molecule that can bind abinding partner and the binding partner is linked to a signal producingagent. For example, in some embodiments, the detectable label is biotinand the binding partner is streptavidin or vice versa. In someembodiments the detectable label is digoxigenin and its binding partneris anti-digoxigenin or vice versa. In some embodiments, the detectablelabel is 2, 4-dinitrophenol (DNP) and the binding partner is anti-DNP orvice versa. Other labels known to one skilled in the art can also beused in the nucleic acid probe disclosed herein. The signal—producingagent can be any agent that produces quantifiable signal, including butnot limited to, chemiluminescence, color, or fluorescence. Non-limitingexamples of signal-producing agents include an enzyme, a fluorescentmolecule, or the like. Other non-limiting examples of detectable labelsand signal-producing agents can be found in US20130209990, herebyincorporated by reference. In one preferred embodiment, thesignal-producing agent is horseradish peroxidase (HRP).

In some cases, the 3′ end of the nucleotide sequence of the probe isconjugated to one or more detectable label. In some cases, both the 5′and 3′ ends of the nucleotide sequence are conjugated to one or moredetectable label. The number of detectable labels conjugated to theprobe can vary, for example, the number can be at least one, two, three,four, five, six, seven, ten, fifteen, eighteen, or more. In general, ahigher number of detectable labels will produce a higher signal in theassay (Division et al., Nucleic Acids Res. (1988) 16: 4077-4095). One ofordinary skill can readily determine the number of detectable labels tobe used based on the amount of signal required to detect the target,e.g., the Alu repeats, in the cell-free DNA from biofluid.

In one embodiment, the 5′ or the 3′ end of the nucleic acid sequence ofthe probe is conjugated to one biotin. In one embodiment the totalnumber of biotin conjugated to the nucleic acid sequence is 1, or 18, orany number in between. The one or more biotin can be all conjugated atthe same 5′ or 3′ end of the nucleic acid probe. The one or more biotincan also be conjugated at both ends, in any combination. In someembodiments, two biotins are conjugated to the nucleic acid sequence ofthe probe. In one embodiment, the two biotins are conjugated to the 5′end of the nucleic acid sequence of the probe.

The nucleic acid probes described herein may be produced by any suitablemethod 3 known in the art, including for example, by chemical synthesis,isolation from a naturally-occurring source, recombinant production andasymmetric PCR (McCabe, 1990 In: PCR Protocols: A guide to methods andapplications. San Diego, Calif., Academic Press, 76-83). It may bepreferred to chemically synthesize the probes in one or more segmentsand subsequently link the segments. Several chemical synthesis methodsare described by Narang et al. (1979 Meth. Enzymol. 68:90), Brown et al.(1979 Meth. Enzymol. 68:109) and Caruthers et al. (1985 Meth. Enzymol.154:287), which are incorporated herein by reference. Alternatively,cloning methods may provide a convenient nucleic acid fragment which canbe isolated for use as a promoter primer. A double-stranded DNA probecan be first rendered single-stranded using, for example, conventionaldenaturation methods prior to hybridization to the target in cfDNA inbiofluids.

5. Method of Use

In some embodiments, the methods involve linking cfDNA (not previouslyamplified) to a solid support, hybridizing a probe specific for an Alurepeat to the cfDNA, removing (e.g., washing away) unbound probe, andthen detecting the amount of specifically hybridizing probe, therebydetermining the amount of cfDNA in a sample.

a. Obtaining Biofluid Samples

Biofluid samples, e.g., urine or BAL, from individuals, e.g., thosesuspected of having organ injury, can be collected in any mannerrecommended by a medical professional, e.g., being collected,mid-stream, in sterile containers. In some embodiments, the sample isthen processed through centrifugation to remove cellular components,thereby producing a cell-free sample. Optionally, the sample can bemixed with a buffer (e.g., PBS or Tris) and/or the above-describedpreservation cocktail. The sample can be stored at −80° C. until furtheranalysis. In some embodiments, a preservative cocktail as describedabove is added to the biofluid sample to produce a mixture. In someembodiments, the mixture can be aliquoted for extraction of cfDNA.

b. Extracting Cell Free DNA

Methods for extracting cell free DNA are well known in the art andcommercial kits are readily available, for example, QIAamp® CirculatingNucleic Acids Kit from Qiagen (Valencia, Calif.). In general, the samplecan be treated to degrade cell debris and remove DNase and RNase toproduce a lysate. DNA in the lysate can be extracted by e.g., passingthe lysate through a DNA binding column and the bound DNA can be elutedwith water or buffer. Optionally, an aliquot of the eluent can be takento determine the concentration of the cfDNA recovered.

The cfDNA is subsequently linked to a solid support. Solid supports canbe a containing vessel, a bead or any other solid support. In someembodiments, the cfDNA is placed in a desired vessel, for example, inthe wells of a microwell plate and incubated for a period of time thatis sufficient to allow the cfDNA to bind surface of the wells. In someembodiments, the incubation occurs over a period of at least one, atleast two, at least four hours long, optionally at room temperature. Insome embodiments, the incubation occurs at 4° C. for at least 8 hours.After the incubation, the vessel can be washed and blocked with ablocking solution, e.g., a solution comprising 5% BSA to minimizenon-specific binding. The blocking solution can comprise a buffer (e.g.,PBS). The blocking solution can then be removed before adding thenucleic acid probe for hybridizing with the target, i.e., the Alurepeats.

c. Hybridizing a Nucleic Acid Probe with Alu Repeats

Hybridization assays of the nucleic acid probe with the Alu repeats incfDNA can be performed in any reaction vessel, including but not limitedto a multi-well plate, e.g., a 96-well plate, e.g., the 96-well LUMITRAC600 (Greiner Bio-One). The assay of detecting cfDNA using ahybridization assay, i.e., hybridization of a probe conjugated to adetectable label without previous amplification of the target nucleicacid is desirable compared to a PCR-based approach because hybridizationassays are cost-effective and can be multiplexed to a higher grade(e.g., 384/batch). In addition, the hybridization approach allows formore complete and accurate quantification of cfDNA as the probe willdetect all single and double-stranded cfDNA in the biofluid,irrespective of fragment length.

In an exemplary hybridization assay, a pre-determined volume of cfDNAextracted from the biofluids as described above is allowed to bind to asupport, e.g., the bottom of the microplate wells. In some embodiments,the surface of the support is previously treated to increase the bindingaffinity of the cfDNA to the surface of the reaction vessel. In someembodiments, a buffer (e.g., PBS) and salts (e.g., MgCl₂) is added tofacilitate hybridization between the probe and cfDNA. The final workingconcentration of the salt can be 0.05M-0.5M, e.g., 0.05M-0.2M, or 0.1M.Optionally, a standard curve can be created using known quantities ofhuman DNA extract.

Hybridization of the cfDNA and the nucleic acid probe can be conductedunder standard hybridization conditions. Reaction conditions forhybridization of a probe to a nucleic acid sequence vary from probe toprobe, depending on factors such as probe length, the number of G and Cnucleotides in the sequence, and the composition of the buffer utilizedin the hybridization reaction. Moderately stringent hybridizationconditions are generally understood by those skilled in the art asconditions approximately 20° C.-50° C., e.g, 25° C.-40° C. below themelting temperature of a perfectly base-paired double stranded DNA.Higher specificity is generally achieved by employing incubationconditions having higher temperatures, in other words more stringentconditions. Chapter 11 of the well-known laboratory manual of Sambrookat al., MOLECULAR CLONING: A LABORATORY MANUAL, second edition, ColdSpring Harbor Laboratory Press, New York (1990) (which is incorporatedby reference herein), describes hybridization conditions foroligonucleotide probes in great detail, including a description of thefactors involved and the level of stringency necessary to guaranteehybridization with specificity. Hybridization is typically performed ina buffered aqueous solution, for which conditions such as temperature,salt concentration, and pH are selected to provide sufficient stringencysuch that the probes hybridize specifically to their respective targetnucleic acid sequences but not any other sequence.

Generally, the efficiency of hybridization between the nucleic acidprobe and target, e.g., the Alu repeats in cfDNA, improves underconditions where the amount of probe added is in molar excess to thetemplate. In some embodiments, the nucleic acid probe of the inventionis diluted in 5% BSA at a concentration of between 10 ng/μl-200 ng/μl,e.g., 20 ng/μl-100 ng/μl, 30 ng/μl-75 ng/μl, 30 ng/μl-50 ng/μl. In aparticular embodiment, nucleic acid probe is the double-biotinylated andis used at the concentration of 30-40 ng/μl. The nucleic acid probe andthe cfDNA in the plate can be incubated to allow the hybridization ofthe probe and the Alu repeats in the cfDNA. The wells of the plate canthen be washed (e.g., with PBS or another buffer) and optionally driedbefore detection.

d. Detecting Signal

Methods of detecting signal produced by detectable labels are well-knownin the art and the methods vary depend on the nature of the chemicalreaction employed to produce the signal. As described above, in someembodiments, the detectable label itself is a signal producing agentthat produce a signal, which can be read directly using appropriateequipment, for example, a plate reader. In some embodiments, thedetectable label produces signal indirectly, a solution comprising abinding partner of the label that is conjugated to a signal producingagent is added to the plate to produce the signal. The signal producingagent include, for example, enzyme or enzyme substrates, reactivegroups, chromophores such as dyes or colored particles, luminescentmoieties including a bioluminescent, phosphorescent or chemiluminescentmoieties, and fluorescent moieties. In one embodiment, the detectablelabel is biotin and the binding partner is streptavidin and the signalproducing agent is horse radish peroxidase (HRP). In this particularembodiment, the signal is a chemiluminescent signal, which can bereadily detected and quantified by methods well known in the art.

e. Detecting Creatinine Levels

The quantification of cfDNA disclosed herein can be combined withmeasurement of a urine protein. In one specific embodiment the urineprotein is creatinine. Creatinine can be measured using the Jaffereaction, an absorbance based method. Commercial assays for measuringcreatinine are readily available, such as the QuantiChrom™ CreatinineAssay Kit (BioAssay Systems), which produces an output in mg ofcreatinine/deciliter of urine. See, e.g.,www.bioassaysys.com/Datasheet/DICT.pdf. In some embodiments, urinecreatinine measurements are taken before or after extracting andquantifying cfDNA in the urine sample. In some embodiments, urinecreatinine measurements and cfDNA quantification are performed in thesame microwell plate (but different wells), by e.g., placing urinesamples for measuring creatinine in the same microplate as the cfDNAsextracted from these urine samples. Measuring the creatinine and cfDNAin the same plate/assay saves time and cost as compared to conventionalPCR methods, which requires one assay to read the amount ofamplification product of the cfDNA and one assay to read creatininelevel.

f. Detecting Additional Markers.

Additional known markers can be measured to further improve thesensitivity and/or specificity of detection of organ injury. In someembodiments, the additional markers comprise one or more tissueinflammatory markers, e.g., CXCL10. e.g., inflammation in the kidney. Insome embodiments, CXCL10 is detected and quantified. Methods formeasuring CXCL10 is well known, for example, using a sandwich ELISAapproach, where a capture antibody adsorbs onto the plate first and thensample is added to allow the CXCL10 in the sample to be captured.Afterwards, a detection antibody is added that will bind the capturedCXCL10. This can be detected using the usual secondary antibody—HRPconjugation approaches well known in the literature. A standard curvewould be generated with standard concentrations of purified CXCL10protein. Commercial kits are readily available for measuring CXCL10, forexample, Human CXCL10/IP-10 Quantikine ELISA Kit from R& D systems.Urine CXCL10 can be measured before or after the cfDNA measurement.Urine CXCL10 can also be measured in the same microwell plate as cfDNA,by e.g., coating CXCL10 capture antibody in designated wells in themicroplate first and placing urine samples for measuring CXCL10 in thesedesignated wells.

In addition, differential methylation status of the cfDNA in urine addsadditional significance for kidney injury and markers correlated withmethylation status can be measured and added to the assay describedabove for detecting kidney injury. Thus, in some embodiments, theadditional markers comprise one or more DNA methylation markers. In oneparticular embodiment, the DNA methylation marker is 5-emthylcytosinethat is incorporated into the nucleic acid in the sample.

DNA methylation markers can be detected and quantified using anELISA-based approach. Commercial kits that are used to measure the DNAmethylation markers are readily available, for example, the MethylFlashUrine 5-Methylcytosine (5-mC) Quantification Kit from EpiGentek that isused to measure the amount of 5-methylcytosine incorporated in the DNA.Briefly, a plate that has methylated DNA is incubated with the sampleand an antibody that recognize the methylation marker. This solution isallowed to incubate and is then washed and further detected with adetection antibody and/or substrate.

In some embodiments, the additional markers comprise total protein inthe biofluid sample. The amount of total protein can be measured usingany methods known in the art that can be used to measure the protein. Inone embodiment, the assay to measure total protein is a colorimetricassay, e.g., the Bradford protein assay.

In some embodiments, the additional markers comprise clusterin.Clusterin is a protein that is associated with the clearance of cellulardebris and apoptosis. The presence of clusterin can also be detected andmeasured using a ELISA-based assay. such as the Human Clusterin DuoSetELISA. Like other sandwich ELISAs, a plate with capture antibody againstClusterin bound to it is incubated with urine samples, optionallydiluted in sample diluent. A detection antibody also against Clusterinis then incubated with the plate, and an HRP-detection system is used tomeasure absorbance.

g. Optional Reagents and Devices.

The methods may be used in a variety of assay devices and/or format. Theassay devices may include, e.g., assay plates, cartridges, multi-wellassay plates, reaction vessels, test tubes, cuvettes, flow cells, assaychips, lateral flow devices, etc., having assay reagents (which mayinclude targeting agents or other binding reagents) added as the assayprogresses or preloaded in the wells, chambers, or assay regions of theassay module. These devices may employ a variety of assay formats forspecific binding assays, e.g., immunoassay or immunochromatographicassays. Illustrative assay devices, e.g., microwell plates and formats,96-well plate format, are described herein below.

In certain embodiments, the methods can employ assay reagents that arestored in a dry state and the assay devices/kits may further comprise orbe supplied with desiccant materials for maintaining the assay reagentsin a dry state. The assay devices preloaded with the assay reagents cangreatly improve the speed and reduce the complexity of assaymeasurements while maintaining excellent stability during storage. Theassay reagents may also include substances that are not directlyinvolved in the mechanism of detection but play an auxiliary role in anassay including, but not limited to, blocking agents, stabilisingagents, detergents, salts, pH buffers, preservatives, etc. Reagents maybe present in free form or supported on solid phases including thesurfaces of compartments (e.g., chambers, channels, flow cells, wells,etc.) in the assay modules or the surfaces of colloids, beads, or otherparticulate supports.

In some embodiments, the methods described above can be performed in alateral flow assay (“LFA”). LFA depends on the capillary action of afluid sample drawn across a pad that contains capture reagents to theantigen of interest. In some embodiments, the biofluid sample is mixedupon application to a device, e.g., a dipstick or a test strip, withreagents that are either purified antigen of interest bound to a visiblemarker (e.g. colloidal gold) in a competitive format or an antibody thatis bound to a visible marker and recognize an antigen of interest in thebiofluid sample in a non-competitive format. The antigen of interest canbe any molecule in the biofluid sample, for example, any of the markersdisclosed in this application, such as creatinine, cfDNA, methylationmarkers, CXCL10, and/or clusterin. In some embodiments, a competitiveformat of LFA is used to measure the creatinine and/or methylationmarkers. In some embodiments, a non-competitive format LFA is used tomeasure cfDNA, CXCL10, and/or clusterin.

6. Determination of Organ Injury Status

a. Determining Organ Health Based on the Amount of cfDNA

In some embodiments, the determination organ health of an individualcomprises comparing the amount of cfDNA in the biofluid sample to acutoff value or predictive probability estimate indicative of organinjury status. The cutoff value can be a pre-determined value orpredictive probability estimate, e.g., a value recommended by medicalprofessionals. Depending on circumstances, it may be necessary in somecases to establish a cutoff value for the determination. To establishsuch a cutoff value for practicing methods disclosed herein, a group ofhealthy individuals, such as a group of individuals who do not haveorgan injury after a organ transplantation is selected. Theseindividuals are within the appropriate parameters, if applicable, forthe purpose of determining organ injury status using the methods of thepresent invention. For instance, the individuals may be of similar age,gender, and comparable health status.

In some embodiments, to assess kidney injury, the detected amount ofcfDNA, i.e., the amount of DNA hybridized to the nucleic acid probe, inurine is first normalized to the amount of urine creatinine to produce anormalized amount of cfDNA. In some cases, the normalized amount ofcfDNA is a ratio of the detected amount of cfDNA to the amount ofcreatinine in urine. To determine the kidney injury status, thenormalized amount of cfDNA is compared to a cutoff value, which is alsoa relative ratio of the cfDNA amount to the creatinine amount in urine,as determined to be indicative of kidney injury status by medicalprofessionals or established as described above.

If the amount of cfDNA or the normalized amount of cfDNA in urine ishigher than its respective cutoff value, the patient is determined tohave kidney injury; in general, a higher value indicates a higher degreeof kidney injury. In some cases, if the amount of cfDNA or normalizedamount of cfDNA is higher than but near the cutoff value, the patient isdetermined to have subclinical injury. If the amount of cfDNA or thenormalized amount of cfDNA in urine is equal to or lower than itsrespective cutoff value, the patient is determined to have no kidneyinjury, i.e., good kidney health. For patients who have received kidneytransplants, detection of kidney injury from urine indicates they arelikely to have acute rejection episodes. For patients who are suspectedof having certain kidney disease, in some embodiments, detection ofkidney injury indicates the patients are likely to have the kidneydisease.

In contrast to conventional methods of detecting cfDNA in the blood,which would be contaminated with cfDNA mostly from the recipient of thekidney transplant, cfDNA detected in the urine specifically reflectsdonor-derived DNA, even when testing for total cfDNA. FIG. 6 shows thecorrelation between chromosome Y and chromosome 1 copy number in theurine. The strong linear correlation (R²=0.9253) indicatesquantification of total cfDNA reflects the donor-derived burden andcorrectly reflects the kidney injury status due to the kidneytransplantation.

b. Determining Organ Injury Status for Organ Transplantation PatientsUsing a IT Score

In certain embodiments, a predictive score, i.e., IT score, is used todiagnose whether a patient who has received an organ transplant may haveorgan injury, or to predict the likelihood a patient will develop organinjury in the future. Organ injury developed after organ transplant istypically associated with acute rejection episodes to the transplantedorgan.

The IT score can be a composite value that can be calculated based onthe amount of cfDNA in a biofluid sample from the organ and a factor(‘normalizing factor”) that can be used to normalize the amount of cfDNAin the biofluid sample. In one embodiment, the amount of creatinine isused to normalize the cfDNA in urine. In another embodiment, the samplevolume of the BAL is used to normalize the cfDNA in BAL from lung. TheIT score may also include the time point, i.e., dayspost-transplantation, when the biofluid sample is taken. The timepost-transplant can be a confounder of organ injury because patientsoften experience injuries that are not necessarily due to the rejectionto the transplanted organ, for example, the ongoing nephrotoxic injuryto the transplanted organ from ischemia reperfusion and nephrotoxicdamage due to infections and calcineurin inhibitor drug exposure. Theseinjuries are not indicative of acute rejection and the extent of suchinjuries may vary at different time points post transplantation. Thus,the IT score, taking the time includes time point post transplantation,can accurately predict whether the patient has acute rejection episodesto the transplanted organ.

In some embodiments, measurements of cfDNA in the biofluid sample may becombined with other biomarkers in the biofluid sample to form the ITscore that can be used to diagnose and/or predict organ injury. In someembodiments, multivariate methods can be used to incorporate these otherbiomarkers, e.g., to CXCL10 and DNA methylation markers, to calculatethe IT score. In some embodiments, cfDNA concentrations are normalizedrelative to the amount of creatinine in the sample by taking the ratioof cfDNA to creatinine or scaling the logarithmic measurements of cfDNAand creatinine proportionately (e.g. through regression analysis). Insome embodiments, cfDNA concentrations are normalized relative to thesample volume of BAL by taking the ratio of cfDNA to the volume orscaling the logarithmic measurements of cfDNA and volume proportionately(e.g. through regression analysis). In some cases, the generated valuesare regressed using generalized linear models incorporating aquasibinomial distribution and a logistic link function. In some cases,the resulting model probability estimates for acute rejection arerescaled from 0-100 to form the IT score and used to generatepredictiveness curve. FIG. 12 shows an illustrative embodiment, whichdisplays the probability of acute kidney rejection in kidney transplantpatients as a function of the resulting a IT score for assessing kidneyinjury (“KIT”).

In some embodiments, an IT score, e.g., a KIT score, can be used topredict or diagnose organ injury with high specificity and sensitivity.In some embodiments, the IT score is generated by including the amountof cfDNA, the amount of DNA methylation markers, and/or inflammationmarkers present in the biofluid sample from the organ that is suspectedof having injury or being likely to develop injury in the future. Insome embodiments, the IT score is a KIT score for assessing kidneyinjury. In some embodiments, the KIT score is generated by furtherincluding the amount of creatinine and/or the amount of a kidney tubularinjury marker (e.g., clusterin). In some embodiments, the IT scoreproduced by including multiple markers as described above can detectorgan injury with a sensitivity of at least 85%, at least 87%, at least88%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, and/or a specificity of at least at least 85%, atleast 87%, at least 88%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%. In some embodiments, using theKIT score described above can detect kidney injury with a AUC of atleast 85%, at least 87%, at least 88%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96.9%, orat least 99.4%. FIG. 13 provides an illustrative embodiment, whichdisplays a IT score for assessing kidney injury (“KIT”) based on adataset of 490 clinical patient samples with multiple causes of kidneyinjury. The resulting KIT score is based on a subset of 299 clinicalpatient samples that had complete measurements of cfDNA, creatine,CXCL10, Clusterin, Protein, and DNA methylation markers; 111 type IIdiabetes mellitus, 71 immune, 50 kidney stones, 20 transplantrejections, 19 hypertension and 28 controls. The resulting KIT score hasover 91% sensitivity and specificity for detection of kidney injury(AUC=96.9%).

In some embodiments, mathematical models/algorithms are used to developthe IT scores using the amount of cfDNA and one or more markers asdescribed above. Such models may include generalized linear models, suchas logistic regression; and nonlinear regression models, such as neuralnetworks, generalized additive models, similarity least squares, andrecursive partitioning methods. In some embodiments, the mathematicalmodels/algorithms that are used to generate the IT score are executed byone or more computer processors. Preferably, the mathematical modelsused to develop the IT scores are based on a database comprisingsufficient sample size. In some embodiments, the database comprises atleast 50, at least 60, at least 70, at least 100, at least 200, at least300, or at least 400 samples. FIGS. 14a-14e provide an illustrativeembodiment of using generalized linear model fits and associated ROCcurves to provide the probability of kidney injury, as well as theprobability of kidney injury due to each disease cause; namely, type IIdiabetes mellitus, immune response, kidney stones, transplant rejection,and hypertension. In one specific embodiment as shown in FIGS. 14A-E,the resulting algorithm has over 92% sensitivity and specificity(AUC>99.4%) for detection of each cause of kidney injury.

In some embodiments, a cutoff value for the IT scores can be establishedby measuring markers present in biofluid samples from the same orsimilar types of organs from a group of healthy individuals, such as agroup of individuals who do not have organ injury after an organtransplantation is selected. These individuals are within theappropriate parameters, if applicable, for the purpose of determiningorgan injury status using the methods of the present invention. Forinstance, the individuals may be of similar age, gender, and comparablehealth status. The cutoff value of the IT scores is then produced usingmathematical models and/or markers that are the same as those used togenerate the IT scores for patients to be tested.

In some embodiments, multivariate methods have been used to incorporatemultiple biomarkers, e.g., to CXCL10 and DNA methylation markers, withcfDNA and creatinine to further refine the KIT score and the KIT scorecan be used to diagnose and/or predict kidney injury that has beeninduced by multiple clinical conditions such as diabetes I, diabetes II,kidney stones, cancer, and immune complexes such as IgA nephropathy.

A number of ways can be used to produce the KIT score. In someembodiments, a cutoff value of the cfDNA/creatinine ratios is firstdetermined based on normalized cfDNA values (e.g., normalized tocreatinine levels) from urine samples from kidney transplant patientswho have not shown rejection. These urine samples can be collected atpredetermined time points over a post-transplantation period. KIT scoresfor a patient can then be determined based on the patient's normalizedcfDNA values. Such normalized values can then be compared to cutoffvalues for those time points to determine whether the normalized valuesare predictive of healthy or diseased kidney function. Such comparisonscan be performed on a computer if desired.

In some embodiments, the cutoff value is the cfDNA/creatinine ratios inpatients who received kidney transplantation but who have not shownorgan injury at respective time points in a post-transplantation period.In some embodiments, a prediction band of normalized cfDNA levels can begenerated and a patient's actual normalized cfDNA value can be comparedto the value in the prediction band corresponding to the same time pointpost-transplantation. The prediction band can be established in avariety of ways. In some embodiments, a number of stable, non-rejectingpatients were examined over time and an exponential decay curve was fitto the cfDNA/creatinine values with respect to time post-transplant. Theprediction band was subsequently generated based on a prescribedprobability to cover the values of future observations from the samegroup that was sampled. For example, a 95% prediction band consists ofupper limits of cfDNA/creatinine ratios from 95% of stable non-rejectingpatients examined at various time points post-transplantation. The KITscore at a particular time post-transplantation is then determined basedon the actual measurements of cfDNA/creatinine and the prediction bandvalue corresponding to that time point.

In some approaches, the cfDNA values relative to the creatinine valuesare processed into other forms of information, e.g., by using eithercommon mathematical transformations such as logarithmic transforms, orstatistical models, such as logistic or generalized linear models. Otherdata processing approaches, such as normalization of the results inreference to a population's mean values, etc. are also well known tothose skilled in the art and can be used.

The KIT score is typically a numerical score on a defined scale orwithin a defined range of values. The KIT score can be compared with acutoff value of the KIT scores that is predictive of whether the patienthas acute rejection episodes. If the KIT score is above the cutoff valueof the KIT scores, a patient is predicted to have kidney injury,indicating he or she is likely to have acute rejection (AR) episodes. Insome cases, a higher KIT score indicates a higher degree of injury dueto the AR episodes. For example, in the case where the KIT score is thelogarithm of the patent's cfDNA/creatinine ratio divided by the cutoffvalue of the ratio, the cutoff value of the KIT score is 0.

The KIT score is highly predictive of organ injuries associated withacute rejection and can be used to determine if the patient has acuterejection episodes. See FIG. 5A. Receiver operating characteristiccurves (ROC) analysis of showed that AUC of ROC curve for detectingacute rejection using the KIT score was 0.9649, with a p-value of 0.0001and a 95% confidence interval of 0.9346-0.9952, indicating the method ishighly sensitive and specific FIG. 5B. In contrast, the conventionalkidney injury prediction method, i.e., detecting proteinuria, had a muchlower AUC of 0.6498, with a p-value of 0.07479 and a confidence intervalof 0.5032-0.7963. The significance of difference was tested usingMcNemar's Paired chi-square from results from the same set of patients.The p-value was 0.021, suggesting that KIT is providing informationabove and beyond protein and that they are significantly different withrespective to sensitivity. This comparison suggests that KIT method ismore acute than the proteinuria method in determining acute rejection

McNemar Test Results:

KIT & Protein Protein KIT .00 1.00 .00 34 20 1.00 7 17 TestStatistics^(a) KIT & Protein N 78 Chi-Square^(b) 5.333 Asymp. Sig. .021^(a)McNemar Test ^(b)Continuity Corrected

In some embodiments, the KIT score is generated by further includingmeasurements of other known kidney injury markers, in addition to thecfDNA/creatinine ratios, at various time point post-transplantationincrease assay sensitivity. These markers include but not limited toCXCL10 and DNA methylation markers, as described above. For anybiomarker of interest, a longitudinal trend curve can be generated foreach of these biomarkers and an exponential decay curve i.e. a 90% or95% predication band, can be established in a manner similar to what isused to generate the prediction band for the cfDNA/creatinine ratios.

Approaches similar to what is disclosed above, i.e., methods ofgenerating a KIT score and using the generated KIT score to assesskidney injury, can be used to produce KIT scores to assess injury of anyother organ, e.g., injury developed after an organ transplant.

Thus, the present invention can be used to conveniently monitor organtransplant patients for organ injuries associated with acute rejectionepisodes by either using the cfDNA normalized amounts (e.g., normalizedto creatinine levels) or the IT scores corresponding to designated timepoints over a period of time post-transplantation. The period can be ofany length as deemed necessary by the treating physician, e.g., at least20 days, at least 50 days, at least 100 days, at least 150 days, atleast 200 days, or at least 400 days, at least 500 days, or the lifetime of the transplanted kidney. Measuring cfDNA and creatinine can beperformed at any frequency as deemed necessary, e.g., at least onceyear, at least twice a year, at least every three months, at least everytwo months, or at least every one month, or at least every 20 days, orat least every 10 days. Patients who are so determined to have acuterejection episode can be treated as soon as possible, for example, byadministering immunosuppressant drugs. Non-limiting examples ofimmunosuppressant drugs include calcineurin inhibitors, such asTacrolimus and Cyclosporine; anti-proliferative agents, such asMycophenolate Mofetil, Mycophenolate Sodium and Azathioprine; mTORinhibitors, such as Sirolimus, steroids such as Prednisone, andinduction agents such as thymoglobulin, IL2R blockade or belatacept. Onthe other hand, if the IT score is equal to or below the cutoff value,the patient is determined to have no kidney injury and thus nointervention is needed. If a patient has a IT score near the cutoffvalue, the patient is determined to have subclinical injury. The ITscores can assist the treating physicians to determine whether thekidney transplantation was successful and whether and when interventionis needed. In addition, a patient specific trend of the IT score canalso be analyzed to determine whether any clinical intervention isneeded. For example, a trend of increase in the IT scores suggests thatthe patient is developing acute rejection episodes and therefore theclinical intervention may be necessary.

c. Detecting Kidney Diseases

In the cases of kidney injury associated with kidney diseases, the KITscore is not applicable, and the determination is based on thecfDNA/creatinine ratio as described above. In some cases, the additionof other biomarkers, e.g., CXCL10 that can be multiplexed onto the cfDNAplate may be applicable in creating a predictive model for thedetection. The methods can be used to detect kidney diseases such asfocal segmental glomerulosclerosis (FSGS), IgA nephropathy, and earlydiabetic kidney disease, BK viral nephritis (BKVN), focal segmentalglomerulosclerosis (FSGS), glomerulonephritis (GN), acute tubularnecrosis (ATN), IgA disease and diabetic kidney disease. FIG. 4C showsthe use of cfDNA/creatinine ratio to separate patients having BKVN fromthose not having the disease.

d. Computer and Smart Phone Devices

In some embodiments, signals from the one or more markers describedherein, e.g., cfDNA, creatinine, e.g., as detected from a lateral flowassay, are transmitted to a computer device, a camera, or a smart phone.In some embodiments, the signals are tranmitted to a smart phone app forprocessing.

EXAMPLES Example 1. The Probe Targeting the Alu Element

To provide more complete coverage of the cfDNA repertoire in thebiofluid, a length of <100 bp were selected for the nucleic acid probetargeting the Alu repeats so that both long and very short fragmentlengths of cfDNA could be captured. In this particular assay, the probelength was 81 bp (FIG. 2), although a shorter length, such as thatcommonly employed by PCR primers of 18-22 bp should work as well.

Reference ALU sequence (SEQ ID NO:1) is listed below and the 81 bp site(SEQ ID NO:2) targeted for the Alu probe is highlighted in bold.

5′GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCA GCACTTTGGGAGGCCGAGGCGGGCGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGT GAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGCGC GCCTGTAATCCCAGCTAC TCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCGGAGGTTGCAGTGAGCCGAGAT CGCGCCA CTGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAA-3′

Biotin was selected due to wide availability of reagents that arecompatible. However, any number of amplification schemes, such asdigioxigenin-anti-digioxigenin antibodies or even direct HRP conjugationwould work.

Dual biotinylated-oligonucleotide complementary to the Alu element wasdesigned and synthesized

(52-Biotin/GCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCGGAGGTTG CAGTGAGCCGAGAT(SEQ ID NO: 2))

This was used to quantify cfDNA using a chemiluminiscence baseddetection system using streptavidin-HRP and chemiluminescent substrate(SuperSignal™ ELISA Femto Substrate) solutions (FIG. 3).

Chemiluminescence was chosen because it is the most sensitive methodavailable in a microwell for HRP detection (FIG. 4), althoughcolorimetric and fluorometric methods will work. Colorimetric methodshave also proved satisfactory. However, colorimetric methods also takelonger to incubate and produce results.

Example 2. Testing the Effectiveness of the DNA Stabilization Solution

In order to test the effectiveness of DNA stabilisation solution inurine containing sonicated genomic DNA, we purified genomic DNA fromwhole blood using QiaAMP DNA Blood Mini Kit (Qiagen), and sonicated itfor 10 min using a Model 550 probe Sonic Dismembrator (ThermoFisherScientific) on intensity setting 3. Cycles were set at 10 seconds on and20 seconds off. Samples were run on 1% gel electrophoresis to establishfragment sizes approx. 150 to 250 base pairs obtained. With sonication,we achieved DNA fragments of approximately 150 to 250 base pairs insize, a range that best represents cfDNA in transplant rejection (FIGS.1A and 1B). Next, we formulated a preservation solution containing 10g/L Diazolidinyl Urea, 20 g/L polyethelyene glycol, 1 mMaurintricarboxylic acid, 10 mM K2EDTA, 10 mM sodium azide, and 1×phosphate buffered saline, pH7.4. An experiment was designed to test theeffectiveness of DNA preservation solution in preserving the integrityof DNA. Fragmented genomic DNA (approx. size-150 to 250 base pairs) wasadded to 1000 μL tubes containing urine with and without preservationsolution. The experiment was run in parallel at 4° C. and at roomtemperature. FIGS. 1A and 1B show-electropherograms that demonstrate howin the absence of DNA preservative solution the DNA degrades anddisintegrates completely over a period of 72 hours, whereas DNApreservation solution helps maintain DNA integrity up to 72 hours.

Example 3. Determine the Acute Rejection Status for Kidney TransplantPatients

a. Sample Collection

Urine samples from three patients (patient #1-#3) having received kidneytransplant were collected, mid-stream, in sterile containers atdifferent time points during the period post transplantation. The urinesamples from patient #1 were taken at day 1, 16, 22, 51, and 180; urinesamples from patient #2 were taken at day 23, day 41, and day 103; andurine samples from patient #3 were taken at day 100, day 132, day 185,and day 337. At a listed time point, in addition to collection of urine,a biopsy is taken to confirm the acute rejection. For example, thelisted time point for patient #1 in this study is day 22. The urinesample was aliquoted into 2 mL for extraction with the QiaAmp®Circulating Nucleic Acids Kit (Qiagen) following the manufacturer'sinstructions with an elution volume of 20 μl in H₂O.

Using a white, opaque ELISA plate such as the 96-well LUMITRAC 600(Greiner Bio-One), 5 microliters of the eluted cfDNA were plated ontothe plate, either in singlicate, duplicate, or triplicate as needed. Tothese sample wells, 10 microliters of a 5×PBS and 0.5 M MgCl₂ bufferwere added and then 35 microliters of molecular grade H₂O was added fora total of 50 microliters per well. To create a standard curve, knownquantities of human DNA extract were added in duplicate in a titrationseries of 1:3 from 200,000 to −12 GE/mL, where a GE (genomic equivalent)is defined as 6.6 pg of human DNA. This was also in the same buffer, afinal working concentration of 1×PBS and 0.1 M MgCl₂.

The cfDNA plate was incubated overnight at 4 C or for a minimum of 2hours at RT shaking at 300 RPM. The liquid was then discarded and theplate was dried via patting against absorbent paper towels. The platewas then blocked in 5% BSA in PBS with 300 microliters per well, for aminimum of 1 hour at RT shaking at 300 RPM. The liquid was thendiscarded and the plate was dried via patting against absorbent papertowels. The plate was then incubated in 50 microliters of ourdouble-biotinylated Alu oligonucleotide probe diluted in the 5% BSA at aconcentration of 35.56 ng/microliters. This was allowed to incubate fora minimum of 1 hour at RT shaking at 300 RPM. The liquid if thendiscarded and then the wells were washed with 300 microliters of 1×PBSthree times. The wash was then discarded and the plate was dried viapatting against absorbent paper towels. The plate was then incubated in50 microliters of streptavidin-HRP diluted 1:200 per manufacturer'sinstructions in 5% BSA and allowed to incubate for no more than 1 hourat RT shaking at 300 RPM. The liquid is then discarded and then thewells were washed with 300 microliters of 1×PBS three times. The washwas then discarded and the plate was dried very thoroughly via pattingagainst absorbent paper towels. 150 μl of SuperSignal ELISA Femtochemiluminescent substrate solution (ThermoFisher) was then added toeach well. The plate was analyzed upon 1 minute of mixing based on totalluminescence.

The generated values were then regressed using a 5-parameter sigmoidcurve fit, such as that provided in GraphPad Prism and the resultantcell-free concentration values were corrected for the dilution done inthe microwell as well as the concentration done from 2 mL of urine to 20μl of eluate.

Creatinine was measured using the QuantiChrom Creatinine Assay Kit(BioAssay Systems) according to manufacturer's instructions.

b. Determining the Cutoff Value of cfDNA/Creatinine Ratio

Prior to the study involving patients #1-#3, cutoff values ofcfDNA/creatinine ratios were established based on data from nine (9)patients, who received kidney transplant and did not have kidney injuryas confirmed by biopsy. cfDNA and creatinine amounts from urine samplesfrom these 9 patients were determined as described herein. The urinecreatinine values were first converted to mg/mL and the cell-free DNAvalues are divided by this creatinine measurement to producecfDNA/creatinine with units of [GE/mg], wherein a GE (genomicequivalent) is defined as 6.6 pg of human DNA. The cfDNA/creatinineratio vs. the days post transplantation was plotted as shown in FIG. 7and a 95% prediction band (the dotted line) as dependent ontime-post-transplant was modeled as an exponential decay curve withfollowing equation.cfDNA/Creatinine[GE/mg]=10{circumflex over( )}((5.612-4.007)*EXP(−0.05977*(Days Post-Transplant))+4.007)/100.

Each data point in this one-sided 95% prediction interval corresponds tothe estimate of the upper limit of the cfDNA/creatinine ratios that,with 95% confidence, is predicted to be higher than cfDNA/creatinineratios from 95% of future non-rejecting patients at the same time pointpost-transplantation. The urine creatinine values were first convertedto mg/mL and the cell-free DNA values are divided by this creatininemeasurement to produce cfDNA/creatinine with units of [GE/mg]. ThecfDNA/creatinine ratio was plotted against days post transplantation.FIG. 8A. KIT prediction scores were calculated as described above, andplotted against days post-transplantation. FIG. 8B. Although between thefirst and second points the cfDNA/creatinine ratio drops (FIG. 8A), theprediction score showed that that relative risk actually increases priorto the biopsy-confirmed acute rejection episode (FIG. 8B) KIT was abovezero at time point 22, indicating that patient #1 had kidney injury andacute rejection episodes. The acute rejection status was confirmed bythe examining the biopsy taken at the listed time point. Patients #1-#3were all given an immunosuppressant after the listed time points:Tacrolimus, MMF, steroids to patient #1, Tacrolimus, MMF, Steroids topatient #2, and Tacrolimus and Sirolimus to patient #3.

cfDNA/creatinine ratios of patients #2-#3 for urine samples were plottedagainst days post-transplantation. FIGS. 9A and 9B. For both patients,acute rejections were predicted using the method disclosed herein at atime point prior to listed time point, when they were confirmed bybiopsy. Additionally, as shown in FIG. 9B, the administration of animmunosuppressant from the listed time point thereon caused the cfDNAratio to decrease into the stable region, indicating successfultreatment Clinical determinants of graft injury can be made on the basisof an absolute elevation in the cfDNA value above the determinedthreshold as well as an increase in the cfDNA burden over time usingpatient specific threshold data. A graft injury is presumed to resultfrom sub-optimal immunosuppression exposure, an abnormal, elevated cfDNAresult could trigger the following clinical actions: 1) return ofpatient to clinic for closer follow-up; 2) consider an earlier protocolbiopsy in a patient scheduled to have one; 3) consider an indicationbiopsy to evaluate for sub-clinical acute rejection and/or other causeof graft injury; 4) a change in immunosuppression drug type or dosing.Conversely, a low, stable cfDNA result could trigger the followingclinical actions: 1) reduce clinic follow-up frequency; 2) avoidunnecessary protocol biopsies that are done to look for sub-clinicalgraft injury; 3) change in immunosuppress ion drug type or dosing.

Example 4. Of Determining the Rejection Status for Lung TransplantPatients

Bronchoalveolar lavage (BAL) fluid samples from 76 patients havingreceived lung transplants were collected in sterile containers eitherduring stable period or during rejection episodes. The BAL fluid wasaliquoted into 400 μL for extraction with the QIAamp Circulating NucleicAcids Kit (Qiagen) following the manufacturer's instructions with anelution volume of 20 μL in H₂O. The cfDNA was measured as disclosed inExample 2.

The generated values were regressed using a 5-parameter sigmoid curvefit, such as that provided in GraphPad Prism and the resultant cell-freeconcentration values were corrected for the dilution done in themicrowell as well as the concentration done from 400 μL of BAL fluid to20 μL of eluate.

The normalized cfDNA concentrations from BAL fluid were compared betweenstable and rejection patients. FIG. 10. The mean level of cfDNA in therejection patients was significantly higher than in the stable patients.An abnormal, elevated cfDNA result could trigger similar clinicalactions as disclosed in Example 2.

Example 5. Determining Kidney Injury Status Using a KIT Score

FIG. 13 provides an illustrative embodiment, which displays a IT scorefor assessing kidney injury (“KIT”) based on a dataset of 490 clinicalpatient samples with multiple causes of kidney injury. The resulting KITscore was based on a subset of 299 clinical patient samples that hadcomplete measurements of cfDNA, creatinine, CXCL10, Clusterin, Protein,and DNA methylation markers; 111 type II diabetes mellitus, 71 immune,50 kidney stones, 20 transplant rejections, 19 hypertension and 28controls. The resulting KIT score had over 91% sensitivity andspecificity for detection of kidney injury (AUC=96.9%). FIGS. 14A-Eprovide results of generalized linear model fits and associated ROCcurves. These results illustrate that in addition to displaying theprobability of kidney injury, the underlying algorithm can accuratelyprovide the probability of kidney injury due to each disease cause;namely, type II diabetes mellitus, immune response, kidney stones,transplant rejection, and hypertension. For these data, the resultingalgorithm showed an over 92% sensitivity and specificity (AUC>99.4%) fordetection of each cause of kidney injury.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentireties for all purposes.

What is claimed is:
 1. A reaction mixture comprising: i) non-amplifiedcell-free DNA (cfDNA) extracted from a urine sample; and ii) a nucleicacid probe having a 3′ end and a 5′ end, wherein the nucleic acid probeis complementary to at least 70 contiguous nucleotides of SEQ ID NO:1and wherein the 3′ end and/or the 5′ end is covalently ligated to atleast one detectable moiety.
 2. The reaction mixture of claim 1, whereinthe detectable moiety is a fluorescent molecule.
 3. The reaction mixtureof claim 1, wherein the detectable moiety is biotin.
 4. The reactionmixture of claim 3, wherein the reaction mixture further comprises astreptavidin-linked signal-producing agent.
 5. The reaction mixture ofclaim 4, wherein the streptavidin-linked signal-producing agent produceschemiluminescence, color, or fluorescence.
 6. The reaction mixture ofclaim 1, wherein the detectable moiety is fluorescein or digoxigenin(DIG).
 7. The reaction mixture of claim 6, wherein the fluorescein ordigoxigenin (DIG) is conjugated to horseradish peroxidase (HRP).
 8. Thereaction mixture of claim 1, wherein the 3′ end or the 5′ end of thenucleic acid probe is covalently linked to at least two detectablemoieties.
 9. The reaction mixture of claim 1, wherein the 3′ and the 5′ends of the nucleic acid probe are covalently linked to at least twodetectable moieties.
 10. The reaction mixture of claim 1, wherein the 3′end or the 5′ end of the nucleic acid probe is covalently linked to atleast three detectable moieties.
 11. The reaction mixture of claim 1,wherein the 3′ and the 5′ ends of the nucleic acid probe are covalentlylinked to at least three detectable moieties.
 12. The reaction mixtureof claim 1, wherein the nucleic acid probe is complementary to 81nucleotides of SEQ ID NO:1.
 13. The reaction mixture of claim 1, whereinthe nucleic acid probe is complementary to at least 70-290 nucleotidesof SEQ ID NO:1.
 14. The reaction mixture of claim 1, wherein the nucleicacid probe is complementary to at least 70-180 contiguous nucleotides ofSEQ ID NO:1.
 15. The reaction mixture of claim 1, wherein the nucleicacid probe is complementary to at least 70-150 contiguous nucleotides ofSEQ ID NO:1.
 16. The reaction mixture of claim 1, wherein the nucleicacid probe is complementary to at least 70-100 contiguous nucleotides ofSEQ ID NO:1.
 17. The reaction mixture of claim 1, wherein the nucleicacid probe is complementary to at least 80-90 contiguous nucleotides ofSEQ ID NO:1.
 18. The reaction mixture of claim 1, wherein the urinesample is from a human who has received a kidney transplant.
 19. Thereaction mixture of claim 18, wherein the urine sample is taken 0-400days, or 10-100 days, or 20-50 days, from the date the human receivedthe kidney transplant.
 20. The reaction mixture of claim 18, wherein theurine sample is taken over the lifetime of the human from the date ofthe human received the kidney transplant.
 21. A method of detecting Alucopy number in cell-free DNA (cfDNA) in a urine sample, the methodcomprising: forming a reaction mixture according to claim 1; andquantifying the amount of cfDNA hybridized to the nucleic acid probe,thereby detecting Alu copy number in cfDNA in the urine sample.